Bruno Lorusso

Sensory neuropathy hampers nociception-mediated bone marrow stem cell release in mice and patients with diabetes

Aims/hypothesisUpon tissue injury, peripheral sensory neurons release nociceptive factors (e.g. substance P [SP]), which exert local and systemic actions including the recruitment of bone marrow (BM)-derived haematopoietic stem and progenitor cells (HSPCs) endowed with paracrine pro-angiogenic properties. We herein explore whether diabetic neuropathy interferes with these phenomena.MethodsWe first investigated the presence of sensory neuropathy in the BM of patients with type 2 diabetes by immunohistochemistry and morphometry analyses of nerve size and density and assessment of SP release by ELISA. We next analysed the association of sensory neuropathy with altered HSPC release under ischaemia or following direct stimulation with granulocyte colony-stimulating factor (G-CSF). BM and circulating HSPCs expressing the neurokinin 1 receptor (NK1R), which is the main SP receptor, were measured by flow cytometry. We finally assessed whether an altered modulation of SP secretion interferes with the mobilisation and homing of NK1R-HSPCs in a mouse model of type 2 diabetes after limb ischaemia (LI).ResultsNociceptive fibres were reduced in the BM of patients and mice with type 2 diabetes. Patients with neuropathy showed a remarkable reduction in NK1R-HSPC mobilisation under ischaemia or upon G-CSF stimulation. Following LI, diabetic mice manifested an altered SP gradient between BM, peripheral blood and limb muscles, accompanied by a depressed recruitment of NK1R-HSPCs to the ischaemic site.Conclusions/interpretationSensory neuropathy translates into defective liberation and homing of reparative HSPCs. Nociceptors may represent a new target for treatment of diabetic complications.

Lung mesenchymal cells function as an inductive microenvironment for human lung cancer propagating cells

OBJECTIVES The aim of the present study was to characterize the biological properties and in vivo tumourigenic potential of mesenchymal cells (MCs) obtained from non-small-cell lung cancer (NSCLC) samples. METHODS NSCLC samples (53 adenocarcinomas and 24 squamous-cell carcinomas) surgically removed from 46 males and 31 females were processed to identify mesenchymal cells from human lung cancer (hLc-MCs). hLc-MCs were separated from neoplastic epithelial cells, expanded and extensively characterized in vitro. Subsequently, female BALB/c nude mice were subcutaneously injected with either 10(6) or 2.5 × 10(6) Calu-3 (human adenocarcinoma cell line able to reproducibly induce xenografted tumours) alone or in combination with equal doses of hLc-MCs. Control animals were injected with the two doses of hLc-MCs only. RESULTS Primary cultures of hLc-MCs were obtained from >80% of NSCLC specimens. The typical MCs immunophenotype was documented by the expression of CD90, CD105, CD73, CD13 and CD44 at fluorescence-activated cell sorting analysis. CD45, CD14, CD34 and epithelial antigens were negative while CD117 (c-kit) and CD133 (prominin) were partially expressed. Interestingly, nuclear transcription factors octamer-binding transcription factor 3/4 and sex determining region Y-box 2 involved in stemness, thyroid transcription factor 1 in bronchoalveolar commitment, and ETS1 in carcinogenesis, were expressed in hLc-MCs isolated from NSCLC. Specific conditioned media and cocultures confirmed the supportive role of hLc-MCs for cancer cells. In vivo experiments showed that at both doses Calu-3 xenografts doubled in size when hLc-MCs were coinjected. Cell tracking in xenografted tumours, by immunofluorescence combined with fluorescence in situ hybridization analysis, documented hX-chromosome-labelled, Calu-3-derived cytokeratin-positive adenocarcinoma structures surrounded by hLc-MCs. CONCLUSIONS Tumour-propagating cells require the inductive interaction of resident mesenchymal cells to foster lung cancer development.

Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy.

Blood and lymphatic vessels contribute to the impact of the immune microenvironment on clinical outcome in non-small-cell lung cancer

OBJECTIVES Lymphangiogenesis plays a critical role in the immune response, tumour progression and therapy effectiveness. The aim of this study was to determine whether the interplay between the lymphatic and the blood microvasculature, tumour-infiltrating lymphocytes and the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint constitutes an immune microenvironment affecting the clinical outcome of patients with non-small-cell lung cancer. METHODS Samples from 50 squamous cell carcinomas and 42 adenocarcinomas were subjected to immunofluorescence to detect blood and lymphatic vessels. CD3pos, CD8pos and PD-1pos tumour-infiltrating lymphocytes and tumour PD-L1 expression were assessed by immunohistochemical analysis. RESULTS Quantification of vascular structures documented a peak of lymphatics at the invasive margin together with a decreasing gradient of blood and lymphatic vessels from the peritumour area throughout the neoplastic core. Nodal involvement and pathological stage were strongly associated with vascularization, and an increased density of vessels was detected in samples with a higher incidence of tumour-infiltrating lymphocytes and a lower expression of PD-L1. Patients with a high PD-L1 to PD-1 ratio and vascular rarefaction had a gain of 10 months in overall survival compared to those with a low ratio and prominent vascularity. CONCLUSIONS Microvessels are an essential component of the cancer immune microenvironment. The clinical impact of the PD-1/PD-L1-based immune contexture may be implemented by the assessment of microvascular density to potentially identify patients with non-small-cell lung cancer who could benefit from immunotherapy and antiangiogenic treatment.